JP2019514387A5

JP2019514387A5 -

Info

Publication number: JP2019514387A5
Application number: JP2018556973A
Authority: JP
Filing date: 2017-04-29
Publication date: 2020-06-18

Claims

A method of assessing the amount of fetus-specific nucleic acid in a sample from a pregnant subject, wherein the sample comprises the fetus-specific nucleic acid and the nucleic acid of interest, the method comprising:
Performing an amplification-based quantitative assay, such as a polymerase chain reaction (PCR) quantitative assay with at least two primer pairs, on a sample or a portion thereof for each of a plurality of single base variant (SNV) targets, Where each primer pair comprises a forward primer and a reverse primer, and one of the at least two primer pairs has a second mismatch from the 3′ end to one allele of the SNV target in the primer, but a different mismatch of the SNV target. Contains a 3'double mismatch and specifically amplifies one allele of the SNV target and the other of the at least two primer pairs specifically amplifies another allele of the SNV target. And
Obtaining or providing results from an amplification-based quantitative assay, such as a PCR quantitative assay, to determine the amount of fetal-specific nucleic acid in a sample,
Including,
Optionally, the method further comprises determining the amount of fetal-specific nucleic acid in the sample based on the result, or the result comprises the amount of fetal-specific nucleic acid in the sample,
The method.

A method of assessing the amount of fetus-specific nucleic acid in a sample from a pregnant subject, wherein the sample comprises the fetus-specific nucleic acid and the nucleic acid of interest, the method comprising:
Obtaining results from an amplification-based quantitative assay, such as a polymerase chain reaction (PCR) quantitative assay, performed on a sample or a portion thereof for each of a plurality of single base variant (SNV) targets, wherein at least 2 Two primer pairs were used, each primer pair including a forward primer and a reverse primer, one of the at least two primer pairs having a second mismatch from the 3'end to one allele of the SNV target in the primers, but A 3'double mismatch to another allele of the SNV target and specifically amplifies one allele of the SNV target, the other of the at least two primer pairs containing the other allele of the SNV target. Specifically amplify, and
Evaluating the amount of fetal-specific nucleic acid based on the results,
Including,
Optionally, the amount of fetal-specific nucleic acid in the sample is based on the results of an amplification-based quantitative assay, such as a PCR quantitative assay,
The method.

Another primer pair of at least two primer pairs also has a second mismatch from the 3'end to another allele of the SNV target in the primer, but a 3'duplex to one allele of the SNV target. Comprising a mismatch and specifically amplifying another allele of the SNV target, and/or the amount is a ratio or percentage of fetal-specific nucleic acids to total nucleic acids,
The method according to claim 1 or 2.

The results are informative results of amplification-based quantitative assays such as PCR quantitative assays,
Optionally, the amount is based on the informative result of an amplification-based quantitative assay, such as a PCR quantitative assay, and/or the method further comprises an informative result of an amplification-based quantitative assay, such as a PCR quantitative assay. Optionally, the selected informative results are averaged, and/or the informative results of an amplification-based quantitative assay, such as a PCR quantitative assay, are fetal-specific nucleic acid and/or Or selected based on the genotype (or parental genotype) of the nucleic acid of interest,
The method according to any one of claims 1 to 3.

The method is more
(I) obtaining the genotype (or parental genotype) of the fetus-specific nucleic acid and/or the nucleic acid of interest;
5. A method according to any one of claims 1 to 4, comprising (ii) obtaining a plurality of SNV targets; and/or (iii) obtaining at least two primer pairs for each of a plurality of SNV targets.

The plurality of SNV targets is at least 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81. , 84, 87, 90, 93 or 96 SNV targets, optionally wherein the plurality of SNV targets is less than 100, 99, 98, 97, 96, 95, 94, 93, 92, 91 or 90 SNV targets. Or the plurality of SNV targets is less than 75 SNV targets, or
The plurality of SNV targets are informative targets, and the plurality of SNV targets are at least 6,7,8,9,10,11,12,13,14,15,16,17,18,19. , 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32 SNVs informative target, optionally, multiple SNV informative targets 34. , 33, 32, 31 or less than 30 SNV informative targets, or more than one SNV informative target is less than 25 SNV informative targets,
The method according to any one of claims 1 to 5.

7. The method of any one of claims 1-6, wherein the amount of fetal specific nucleic acid in the sample is less than 10%, optionally the amount of fetal specific nucleic acid in the sample is less than 5%.

If the parental genotype is unknown or not available, the method further comprises assessing the result based on the prediction of the parental genotype, optionally the assessing is an expected value maximization algorithm. The method according to any one of claims 1 to 7, which is carried out using.

The fetal-specific nucleic acid is fetal-specific cell-free DNA, optionally the amount is used to determine the risk to the fetus, and, optionally, the risk to the fetus is provided herein. 9. The method of any one of claims 1-8, which is a risk of one condition.

10. The method of any one of claims 1-9, wherein the multiple PCR quantitative assays are real-time PCR assays or digital PCR assays.

The method is more
(I) selecting a treatment for the subject based on the amount of fetal-specific nucleic acid;
(Ii) treating the subject based on the amount of fetus-specific nucleic acid;
(Iii) providing information regarding treatment of the subject based on the amount of fetus-specific nucleic acid;
(Iv) monitoring or suggesting monitoring the amount of fetal-specific nucleic acid in the subject over time;
(V) assessing the amount of fetal-specific nucleic acid in the subject at a later time point;
(Vi) assessing the effect of treatment administered to the subject based on the amount of fetal-specific nucleic acid;
(Vii) providing or obtaining a sample or part thereof;
11. A method according to any one of claims 1 to 10, comprising (viii) extracting nucleic acid from the sample; and/or (ix) a pre-amplification step using primers for the SNV target.

12. The method of any one of claims 1-11, wherein the sample is from a subject at least 10 weeks of gestation, and optionally an amount of fetus-specific nucleic acid of less than 10% is indicative of fetal distress. ..

13. The method of any one of claims 1-12, wherein the sample comprises blood, plasma or serum.

A composition or kit comprising:
A primer pair is included for each of the at least 6 informative targets of the SNV, wherein each primer pair contains a second mismatch from the 3'end to one allele of the SNV target in the primer, but a different mismatch of the SNV target. Contains a 3'double mismatch for alleles of and specifically amplifies one allele of the SNV target,
Optionally, the composition or kit further comprises a separate primer pair for each of the at least 6 informative targets of the SNV, wherein the different primer pair specifically amplifies another allele of the SNV target, and /Or the informative target of at least 6 SNVs is at least 7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24. , 25, 26, 27, 28, 29, 30, 31 or 32 SNV informative targets, optionally at least 6 SNV informative targets are 35, 34, 33, 32, 31, Is an informative target of less than 30, 29, 28, 27, 26 or 25 SNVs,
The composition or kit.

For each of the at least 6 informative targets of the SNV, another primer pair also has a second mismatch from the 3'end in the primer to another allele of the SNV target, but to one allele of the SNV target. 15. The composition or kit of claim 14, which comprises a 3'double mismatch and specifically amplifies another allele of the SNV target.

A composition or kit comprising:
A primer pair is included for each of at least 18 SNV targets, wherein each primer pair contains a second mismatch from the 3'end to one allele of the SNV target in the primer, but to another allele of the SNV target. Contains a 3'double mismatch and specifically amplifies one allele of the SNV target,
Optionally, further comprising another primer pair for each of the at least 18 SNV targets, wherein the other primer pair specifically amplifies another allele of the SNV target and/or at least 18 SNV targets , At least 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 71, 75, 80, 85, 90 or 95 SNV targets Optionally, at least 18 SNV targets are less than 100, 99, 98, 97, 96, 95, 94, 93, 92, 91 or 90 SNV targets, or at least 18 SNV targets are 75 Less than SNV target,
The composition or kit.

For each of at least 18 SNV targets, another primer pair also had a 3'-second mismatch in the primer to another allele of the SNV target, but 3'to one allele of the SNV target. 17. The composition or kit of claim 16, which comprises a double mismatch and specifically amplifies another allele of the SNV target.

(I) a buffer (ii) a polymerase (iii) probe, optionally a fluorescent probe,
(Iv) instructions, optionally instructions for determining or assessing the amount of unnatural nucleic acid in a sample,
The composition or kit according to any one of claims 14 to 17, further comprising:

19. Any one of claims 14-18 for use in a method according to any one of claims 1-13 or for use in any one of the methods provided herein. A composition or kit according to paragraph.

Method,
Obtaining an amount of a fetus-specific nucleic acid based on the method according to any one of claims 1 to 13, and evaluating a risk in a subject or a fetus based on the amount.
Optionally, information regarding treatment or treatment or no treatment is selected for or provided to the subject based on the assessed risk; and/or the method further determines the amount of fetal-specific nucleic acid in the subject. Optionally monitoring, or suggesting monitoring, wherein the subject is optionally monitored at one or more time points during the 10th gestation period and beyond,
The method.